The goal of this work is the elucidation of the mechanisms controlling the rate of protein synthesis at the cytoplasmic level in mammalian cells and the differences between these mechanisms in normal and malignant cells. We have demonstrated that, upon nutrient deprivation (essential amino acid, glucose, or serum) of the Ehrlich ascites tumor cell in suspension culture, one such mechanism modulates chain initiation by inhibiting the formation of the complex between the 40s ribosomal subunit and the Met-tRNAf.eIF-2.GTP ternary complex (i.e., the 40s initiation complex). We are now examining eIF-2 function. During the initiation reactions, GTP in the ternary complex is hydrolyzed and eIF-2 is released as, or rapidly forms, eIF-2.GDP. We have found a GDP/GTP exchange factor (GEF) that allows GTP to replace GDP in the binary complex. This is a new initiation factor, since without GEF the half-time of dissociation of the eIF-2.GDP complex is greater than 30 min. Phosphorylation of the alpha subunit of eIF-2 prevents the GEF-catalyzed GDP exchange and thus can stop eIF-2 cycling and chain initiation. Over the past year, we have measured the alterations in the extent of phosphorylation of eIF-2 in cells deprived of an essential amino acid. We find less than 10% change in extent of phosphorylation of total eIF-2 and of the subfraction of eIF-2 bound to ribosomes, bound to free 40s ribosomal subunits, and soluble eIF-2. We are presently developing approaches to determine whether these changes can account for the large changes seen in the rate of protein synthesis. We have also found a dramatic inhibition of eIF-2 function in heat-shocked Ehrlich cells, and we are now measuring eIF-2 phosphorylation in these cells. The defect in eIF-2 function persists in the cell-free protein-synthesizing system prepared from heat-shocked cells. Using the fractionated cell-free system as the assay, we have isolated from non-heat-shocked cells a factor that reverses the defect. (F)